Hyperkalemia is a common condition in chronic kidney disease (CKD) patients because of decreased ability of kidney to excrete K as well as the use of inhibitors of renin-angiotensin-aldosterone system. Paleolithic and Mediterranean diets have low Na, high K contents and are well known to be protective against cardiovascular diseases and CKD. However, it remains unclear how the kidneys handle such a high K load in the context of low Na intake. This is crucial to choosing effective diuretics and preventing the drastic consequences of hypokalemia and hyperkalemia for patients on these diets. For instance, our preliminary findings in this proposal suggest that the K-wasting loop diuretics may become K-sparing and decrease K excretion on low Na high K diet. In the thick ascending limb (TAL), Na+, K+, and Cl- are reabsorbed via Na+-K+-2Cl- cotransporter (NKCC2), and most of the K+ is recycled back into lumen by apical Renal Outer Medullary K channel (ROMK) to facilitate NaCl reabsorption by NKCC2. It has been long understood that the high interstitial K concentration induced by high K diet would inhibit the predominant basolateral K channels, leading to membrane depolarization and inhibition of Cl- exit, and thus inhibit NaCl reabsorption via NKCC2 in the TAL. However, our lab has recently discovered that in mice on a low Na, high K diet (LNaHK), there is a considerable furosemide-sensitive Na+ reabsorption as well as a net K+ secretion in the TAL. Contrary to the traditional view, we have also found that the NKCC2 expression is increased on LNaHK diet. We hypothesize that on LNaHK diet, apical ROMK is upregulated and becomes the predominant K channel which is not affected by high interstitial K+ concentration. NKCC2 is upregulated to supply Na+ to the basolateral Na+-K+ ATPase and bring in more K+ to be secreted. Unlike previous studies, we now have more advanced tools to further investigate the ion transport in TAL such as knockout mice. The hypothesis of this proposal is that the high levels of aldosterone (aldo) and angiotensin II (Ang II) induced by LNaHK diet increase NKCC2 and ROMK activities in the apical membrane of the TAL, thereby causing a net K+ secretion. Two specific aims will investigate the mechanisms that regulate net K secretion in TAL: Specific Aim 1. Determine the role and regulation of NKCC2 in the net K+ secretion in TAL. Specific Aim 2: Determine the role and regulation of ROMK in the net K+ secretion in TAL. We will determine the expression of NKCC2 and ROMK in TAL using western blot and real- time PCR in mice on LNaHK compared to control diet. We will measure the functional activity of NKCC2 and ROMK in TAL using micropuncture, atomic absorptive spectrometry, and patch clamp techniques. We will also perform adrenalectomy (ADX) and use aldosterone (aldo) replacement and inhibitors of renin-angiotensin- aldosterone system to elucidate the regulation of NKCC2 by aldo and Ang II. By fulfilling this proposal, we will have a better understanding of the renal K handling in people on the cardioprotective low Na high K diets.